The goal of our research program is to understand the mechanism of regulation of biological activity in macromolecular complexes through structural analysis, using X-ray crystallography as our primary tool. (1) Adeno-associated virus (AAV) Rep. Only one animal virus, AAV, integrates its genome into a particular location in human chromosomal DNA. This unique property has important implications for the targeted delivery of genes in the context of gene therapy vectors. Viral integration requires the AAV Rep protein, a multifunctional enzyme that possesses site-specific DNA binding activity, endonuclease activity, ATPase activity, and 3'-to-5' helicase activity. To shed light onto the mechanism of integration, we have recently determined the structure of the endonuclease domain of AAV5 Rep bound to the direct repeats that are found both at the Rep binding site at the viral termini and at AAVS1, the site of integration in chromosome 19. We have also determined the structure of the endonuclease domain bound to the stem-loop that occurs on one arm of the three-way DNA junction that marks the end of the viral genome. These structures lead to a model for Rep assembly on the viral termini that we are currently testing using biochemical and structural approaches. (2) DNA transposition. We are interested in the variety of enzymes used by mobile DNA elements to move DNA from one location to another. We are currently investigating two systems: the bacterial Tn7 transposon and Hermes, a eukaryotic transposon from Musca domestica. The Tn7 transposon is unusual as it encodes several protein molecules (TnsA,B,C, and D) necessary for its excision and subsequent integration. To understand the structural basis of transpososome assembly and regulation, we are studying the complexes formed along the transposition pathway and have recently determined the structure of TnsA, the component responsible for 5' end cleavage, bound to the C-terminal region of TnsC, the central regulatory molecule in Tn7. The structure demonstrates that TnsC stabilizes TnsA and aids the binding of TnsA to DNA. Hermes serves as a model system for the hAT family of transposons, representatives of which are found in fungi, plants, and animals including vertebrates. To date, no structure has been reported for any member of the hAT family. We have been successful in crystallizing an active fragment of Hermes, and structure determination is currently underway. (3) Structural biology of complexes of 14-3-3. The 14-3-3 proteins, a family of dimeric regulatory proteins, are found within all eukaryotic cells and are involved in many biological processes. We are continuing our efforts to characterize biologically relevant complexes between 14-3-3 and cellular proteins. We are currently investigating the phosphorylation-dependent interaction between 14-3-3 and the regulatory domain of tyrosine hydroxylase. This enzyme is the rate-limiting step in the production of catecholamine neurotransmitters, and as such is highly regulated. For example, when Ser40 is phosphorylated, there is increased catecholamine biosynthesis, an effect believed to be mediated by 14-3-3. We have confirmed the phosphorylation-dependent binding of 14-3-3 and the regulatory domain using recombinant proteins, and structural studies are in progress.